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Carbohydrate-protein interactions appear to play an important role in the phagocytosis of microbial prey by free-living protozoa. The present study utilizes FITC-labelled plant lectins to investigate the presence and localization of cell surface glycoconjugates on live and fixed planktonic protists (Dunaliella primolecta, Oxyrrhis marina, Goniomonas amphinema, Paraphysomonas vestita and Euplotes vannus). With live flagellate preparations, lectins primarily bound to external cell surfaces, with minimal internal staining observed. In contrast, cell fixation permeabilized cell membranes, allowing lectins to bind to internal structures, such as nuclear membranes and food vacuoles, interfering with the characterization of cell surface glycoconjugates. The method developed to label cell surface sugar moieties of live planktonic protists successfully overcomes the problems associated with fixation, and thus provides a useful protocol for future studies on protistan cell surface carbohydrate characterization.

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

The polymerase chain reaction was used to amplify and partially sequence the 16S ribosomal RNA genes of symbiotic bacteria within the anaerobic ciliate Metopus contortus. In situ probing with fluorescent oligonucleotides showed that the amplified sequences originated from a single species of archaebacterium which is closely related to Methanocorpusculum parvum. The probed symbionts exhibited a variety of shapes and sizes. These data support the hypothesis, first proposed on the basis of electron microscopy, that the symbionts undergo a morphological transformation as part of the symbiotic process.